Preference for IgG mAb binding insulin in solution or on surfaces is related to immunoglobulin variable region structures.
Studies on insulin autoantibodies often show a lack of correlation between enzyme-linked immunosorbent assays (ELISAs) and fluid phase radioimmunoassays (RIAs). Similarly, a set of IgG anti-insulin monoclonal antibodies (mAb) from BALB/c mice are found to differ in their binding in ELISAs and RIAs. To understand the structural basis for differential insulin binding, soluble and complexed biotinylated insulin is used to confirm binding properties independent of insulin-coated plastic and radioiodination. The binding properties of intact mAb are also present in Fab fragments, indicating ligand preference is not related to valence or to the Fc portion of Ig. Analysis of binding to soluble or bound ligand in relationship to antibody variable (V) region structures indicates that differential binding in the two assays is a property of heavy chain variable region structure. Studies also show that limited amino acid replacements arising during maturation of the immune response may change the binding preference for an individual mAb. These findings indicate that differences in detection of insulin binding in solid phase and fluid phase are not artefactual but reflect intrinsic structural features of immunoglobulin interaction with insulin.
Tikhomirov
O Y
OY
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Thomas
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D001710:Biotin; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D007328:Insulin / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010641:Phenotype; D011863:Radioimmunoassay; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013329:Structure-Activity Relationship; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007328:Insulin; D012996:Solutions; D001710:Biotin